TAM receptors in cardiovascular disease

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..

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TAM receptors in cardiovascular disease

Lucy McShane

1,2

,

Ira Tabas

3 ,

Greg Lemke

4,5

,

Mariola Kurowska-Stolarska

6 *, and

Pasquale Maffia

1,2,7

*

1

Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK;2Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK;

3

Departments of Medicine, Physiology, and Cell Biology, Columbia University Irving Medical Center, New York, NY, USA;4

Molecular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA;5Immunobiology and Microbial Pathogenesis Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA;6Rheumatoid Arthritis Pathogenesis Centre of Excellence (RACE), Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK; and7Department of Pharmacy, University of Naples Federico II, Naples, Italy

Received 4 January 2019; revised 28 February 2019; editorial decision 14 March 2019; accepted 9 April 2019; online publish-ahead-of-print 13 April 2019

This article was handled by a Consulting Editor, Ziad Mallat.

Abstract

The TAM receptors are a distinct family of three receptor tyrosine kinases, namely Tyro3, Axl, and MerTK. Since

their discovery in the early 1990s, they have been studied for their ability to influence numerous diseases, including

cancer, chronic inflammatory and autoimmune disorders, and cardiovascular diseases. The TAM receptors

demon-strate an ability to influence multiple aspects of cardiovascular pathology via their diverse effects on cells of both

the vasculature and the immune system. In this review, we will explore the various functions of the TAM receptors

and how they influence cardiovascular disease through regulation of vascular remodelling, efferocytosis and

inflam-mation. Based on this information, we will suggest areas in which further research is required and identify potential

targets for therapeutic intervention.

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Keywords

TAM receptors

_•

Tyro3

_•

Axl

_•

MerTK

_•

Cardiovascular disease

1. Introduction

Insight from the results of studies using cultured cells, mouse models of

cardiovascular disease subjected to genetic engineering or

pharmacolog-ical intervention, and observational studies in humans have provided

strong evidence that the immune responses plays an important role in

cardiovascular pathologies.

1–3

Most importantly, the CANTOS trial

(Canakinumab Anti-inflammatory Thrombosis and Outcomes Study)

has recently demonstrated the efficacy of targeting interleukin (IL)-1b

for reducing secondary cardiovascular events,

4

and therefore,

much of

current research is now focusing on how to limit inflammation to

pre-vent cardiovascular diseases (CVD).

Among the many molecules that influence the immune response, the

TAM family of tyrosine kinase receptors have demonstrated a capacity

to influence the function of both the vascular and immune system in the

steady state and in pathology. Accordingly, there has been much interest

in their potential roles in CVD and how they may be viewed as potential

therapeutic targets. This family of proteins includes Tyro3, Axl, and

MerTK, with the first letters of each giving the family its name.

5

They

remained orphan receptors for the first few years following their

discovery, but by the mid-1990’

s their ligands were identified as growth

arrest-specific 6 (Gas6) and Protein S (Pros1).

6,7

These ligands bind to

the TAM receptors with differential affinity. Gas6 can associate with all

three receptors, but with strongest affinity to Axl, then Tyro3, and with

lower affinity to MerTK.

6

Pros1,

however, does not bind to Axl at all,

and has stronger affinity binding with Tyro3 than MerTK.

8

2. TAM receptors

2.1 Structure

The basic structure of the TAM receptors includes an extracellular

N-terminal region containing two immunoglobulin (Ig)-like domains,

fol-lowed by two fibronectin type III (FNIII) domains, a hydrophobic domain

which traverses the cell membrane, and finally, an intracellular tyrosine

kinase C-terminal domain.

9,10

_{The Gas6 and Pros1 ligands possess an}

ap-proximately 50 amino acid stretch which contains gamma carboxylated

glutamic acid residues, referred to as the Gla-domain. These residues

have a high affinity for calcium, which facilitates binding to

phosphatidyl-serine (PtdSer) molecules found on the surface of platelets and on the

*Corresponding authors. Tel:þ44 141 330 7142; E-mail: [email protected] (P.M.) Tel: þ44 141 330 6085; E-mail: [email protected] (M.K.-S.)

VC_{The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.}

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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outer leaflet of cell membranes under certain conditions, notably on

apoptotic cells.

11

The C-terminus of the ligands possess laminin G

(glob-ular) domains, which facilitate their interactions with the TAM receptor

Ig-like domains.

12

As for most receptor tyrosine kinases (RTKs),

activa-tion of the TAM receptors occurs via ligand facilitated dimerizaactiva-tion,

which mediates autophosphorylation of their tyrosine kinase domain.

9

This results in coupling of the receptor with proteins involved in

signal-l

ing pathways, which will be discussed in more detail below.

2.2 TAM receptor functions

Remarkably, TAM receptors are not required for embryonic

develop-ment, which is unusual for one RTK, let alone an entire subset.

13

However, this property enabled the generation of viable triple TAM

knock-out (KO) mice, which propelled studies exploring the functions of

the TAM receptor family. When these KO mice reach adulthood, three

distinct phenotypes can be observed. The first is male infertility, which

occurs due to an inability to clear apoptotic gamete cells in the testes.

13

The second phenotype is blindness due to retinal epithelial cells not

be-ing able to engulf the outer segments of the photoreceptors, which is

necessary for the removal of toxic byproducts of phototransduction.

14

The third phenotype is autoimmunity.

15–18

This stems from the

require-ment to clear the apoptotic bodies generated during the immune

response in order to resolve inflammation.

19

Failure to clear dead cells

can cause them to become necrotic, with their accumulation serving as a

source of self-antigen.

20

Phagocytosis of apoptotic cells (efferocytosis) is a fundamental

pro-cess for the restoration of immune and tissue homeostasis,

21

and the

TAM receptors play an important role in efferocytosis in adult

tissues.

22,23

In atherosclerosis, efferocytosis is required for clearing of

apoptotic cells in lesions, and in advanced atherosclerosis, this process

can go awry, leading to post-apoptotic necrosis of lesional cells.

24–28

This

pathological process can lead to large areas of plaque necrosis, which are

highly inflammatory and render the plaques susceptible to rupture or

erosion. Ruptured or eroded plaques can then promotes occlusive

vas-cular thrombosis, leading to acute coronary syndromes such as

myocar-dial infarction (MI)

, unstable angina, sudden cardiac death, or stroke.

29

In

atherosclerosis, efferocytosis is largely orchestrated by professional

phagocytes such as macrophages, which are known to express and

uti-lize TAM receptors in this process.

30–32

In other settings, it is likely that

the non-professional phagocytes including cardiac myofibroblasts and

epithelial cells also express TAM receptors,

33,34

particularly Axl which

shows high levels of expression in the heart.

35,36

In addition to the anti-inflammatory effects of efficient efferocytosis,

the TAM receptors are involved in directing the change in the immune

response from attack-the-pathogen to repair-and-restore

(reso-lution).

16,37

This was first indicated by the phenotype of the triple TAM

receptor KO mice, which develop a lymphoproliferative disorder and

broad-spectrum autoimmunity driven by chronic hyper-activation of

an-tigen presenting cells such as monocytes, dendritic cells (DCs),

and

macrophages.

15

Expression of TAM receptors is up-regulated in innate immune cells

upon their activation in order to prime the system for negative feedback,

which is subsequently facilitated by the increased availability of their

ligands upon initiation of the adaptive response.

10

Upon ligand-mediated

autophosphorylation, TAM receptors can physically associate with the

type I interferon receptor (IFNAR)-STAT1 complex, which normally

drives the initial amplification of inflammation. However, the association

between the R1 subunit of IFNAR and the phosphorylated TAM causes

a change in the function of the IFNAR-STAT1 complex to that of an

anti-inflammatory signall

ing molecule, which in turn initiates transcription

of the suppressor of cytokine signalling (SOCS)1 and SOCS3 proteins.

16

These proteins ultimately suppress both cytokine receptor and Toll-Like

receptor (TLR)3, TLR4,

and TLR9 pro-inflammatory signalling

pathways.

38

This relationship between TAM receptors and ligands is key

to maintaining immune homeostasis, particularly between T cells, which

produce Pros1 upon activation, and DCs expressing the TAM

receptors.

39

T cell-DC interactions have been shown to occur within

atherosclerotic lesions, which can enhance the pro-inflammatory nature

of the plaque.

40–42

Therefore, deregulation of TAM-mediated

suppres-sion and resolution of the inflammatory response can strongly influence

cardiovascular immunity.

2.3 Regulation of TAM receptor expression

The expression and activity of the TAM receptors are controlled by

vari-ous factors at the transcriptional, post-transcriptional, and protein levels.

Induction of specific TAM receptor genes can be mediated by various

cytokines. For example, transforming growth factor b (TGF-b), and

granulocyte-macrophage colony-stimulating-factor, and several

proin-flammatory stimuli can drive Axl expression, while IL-17A, IL-10, and

dexamethasone drive MerTK expression.

43–46

As Tyro3 has not been

closely studied in the context of inflammation or the immune response,

it remains unclear which cytokines, if any, specifically up-

regulate its

expression.

Up-regulation of MerTK expression can also be induced by

glucocor-ticoids and the liver-X-receptors (LXR) a and b.

47,48

TAM receptor

reg-ulation by the LXRs is of interest in CVD due to their role in cholesterol

biosynthesis and

homeostasis, notably cholesterol

efflux

from

macrophages.

49

As an example of another level of regulation, microRNA-34a has

been demonstrated to suppress protein expression of Axl, and this

pro-cess can contribute to the autoimmune disorder rheumatoid arthritis.

50

Similarly, microRNA-7 inhibits Tyro3 expression and is consequently

be-ing explored as an RNA-based therapeutic for treatbe-ing abhorrent Tyro3

overexpression in human hepatocellular carcinoma.

51

Once TAM receptors are fully synthesized, inserted into the plasma

membrane, and activated, their extracellular domain can be cleaved by

the metalloprotease A Disintegrin And Metalloproteinase

(ADAM)-17.

52,53

This cleavage liberates a soluble extracellular domain bound to

li-gand (sAxl or sMer) and destroys TAM receptor function. Conversely,

secretory leucocyte

protease inhibitor increases the expression of

MerTK on the cell surface, likely by inhibiting its cleavage.

54

The

molecu-lar regulation of TAM receptors is illustrated in Figure

1 .

3. Axl in CVD

3.1 Gas6-Axl and regulation of

cardiovascular remodelling

Vascular remodelling refers to a dynamic process that causes changes to

the structure of the vascular wall. This can occur in response to certain

stimuli, such as injury or local production of inflammatory mediators. In

addition, chronic conditions such as hypertension or atherosclerosis can

drive the process of vascular remodelling. Although vascular remodelling

likely represents a response to correct environmental changes to

vascu-lar flow and maintain homeostasis, it can have pathological effects, as

de-scribed below. Several vascular cell types, including endothelial cells

(ECs), vascular smooth muscle cells (VSMCs), fibroblasts and

myofibro-blasts, contribute to vascular remodelling. Vascular remodelling occurs

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..

through four main processes: cellular migration, proliferation, survival;

and extracellular matrix synthesis and degradation. For each of these

processes, Axl and its ligands have been shown to play a role, while the

contributions of MerTK and Tyro3 are less clear.

Both Pros1 and Gas6 were shown to be secreted by and enhance the

proliferation of VSMCs prior to their identification as the ligands for

TAM receptors.

58,63

Subsequently, Axl expression was found to be

in-creased in VSMCs following balloon-injury in rat carotid artery, and both

Axl and Gas6 expression were temporally correlated with neointima

formation.

64

The key role of Gas6/Axl pathway in the regulation of

vas-cular remodelling was confirmed in animal studies, which showed

re-duced intimal thickening following vascular injury in Axl

-/-

mice

compared with wild type control mice.

59,65,66

Similarly, in the

deoxycor-ticosterone acetate (DOCA)-salt hypertensive mouse model, Axl

defi-ciency led to reduced systolic blood pressure

67,68

and reduced

remodell

ing index of the mesenteric artery.

68

Emerging studies suggest

Figure 1

Molecular regulation of the TAM receptors. The expression and activity of the TAM receptors is controlled by various factors at the

tran-scriptional, post-trantran-scriptional, and protein levels. (A) TAM receptor gene transcription can be up- or down-regulated by various factors, including

cyto-kines. The figure shows mediators that specifically regulate MerTK transcription; the other TAM receptors are regulated by other mediators.

43,44,46–48

(B)

Post-transcriptional regulation by micro-RNAs such as miR-34a inhibition of Axl expression.

50

_{(C) At the protein level, TAM receptors are rendered}

dys-functional by cleavage of their extracellular domain by metalloprotease ADAM17.

52,53

This process which can be driven by other environmental factors

such as reactive oxygen species (ROS).

53,55,56

_{(D) The soluble byproduct released may act as a decoy for the receptor ligands, thus inhibiting TAM}

recep-tor activity.

57

(E) Activation of the receptors can also be enhanced by various environmental factors.

58–60

(F) Activation of the TAM receptors

subse-quently induces various molecular pathways affecting cell function.

16,60–62

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..

.

that (i) induction of proliferation of VSMCs,

58,63

(ii) stimulation of

migra-tion of VSMCs,

69

and (iii) protection from apoptosis

70,71

are underlying

mechanisms linking Gas6/Axl signall

ing to vasculature remodelling.

Angiotensin II (Ang II) is a hormone produced as an end-product of

the renin–angiotensin system and is an important mediator of numerous

cardiovascular pathologies, such as hypertension, vascular remodelling,

and neointima formation.

72

Ang II has been demonstrated to activate the

Gas6-Axl pathway and is necessary for facilitating the effects of Axl on

VSMC proliferation.

73,74

In a similar vein, reactive oxygen species (ROS)

such as hydrogen peroxide (H

2

O

2

) elicit pathological effects on the

vasculature,

75

at least partially through Axl in VSMCs.

59

ROS have been

shown to induce interaction between Axl and glutathiolated non-muscle

myosin heavy chain (MHC)-IIB, which may mediate increased migration

in vascular injury,

76

and pharmacological inhibition of Axl attenuated the

pathological effects of oxidative stress and reduced VSMC migration

in vitro.

77

In addition, Axl appears to prolong VSMC survival, and Gas6

was shown to protect VSMCs from apoptosis.

70,71

In this context, one

study observed increased VSMC apoptosis in Axl deficient mice, leading

to reduced intima-media thickening following ligation of the left carotid

artery.

66

In addition to regulating VSMC biology, activation of Axl via Gas6

exerted a mitogenic effect on serum-starved fibroblasts in vitro and

pro-tected these cells from cell death by apoptosis.

78

Similarly, Gas6-Axl

signal-ling can promote the survival and vascular endothelial growth factor

A-mediated migration of ECs.

79–83

Finally, Axl has also been shown to directly

regulate cytokine/chemokine expression and extracellular matrix

remodel-ling in the vessel wall.

84

Axl regulates these multiple aspects of vascular

function through its ability to interact with multiple downstream signalling

pathways. For example, Axl-mediated effects on cell proliferation and

migration are facilitated through activation of phosphatidylinositol-3-OH

kinase (PI3K)/protein kinase B (Akt), sarcoma (SRC) signalling pathways,

and extracellular signal-regulated kinases (ERKs), which is similar to other

RTK-mediated processes.

60,61

Remarkably, differing levels of glucose in vitro can affect Axl behaviour,

demonstrated by the comparison of functional Axl interactions in

VSMCs exposed to 5.5 mmol/L ‘low glucose’ or 27.5 mmol/L ‘high

glucose’ culturing conditions.

60

Axl was found to preferentially interact

with proteins involved in the PI3K signalling pathway under ‘low glucose’

conditions, stimulating anti-apoptotic signalling and enhancing survival of

the VSMCs. In contrast, Axl associated with signalling proteins of the

ERK1/2 pathway in ‘high glucose’ conditions, driving VSMC migration.

Ultimately, this demonstrates that the function of Axl may be altered

depending on the physiological conditions.

60

In addition to this, Axl

ex-pression is significantly lower in left internal mammary artery tissue from

diabetic compared with non-diabetic patients

85

and Axl overexpression

was able to reverse the effects of high glucose-induced dysfunction in

ECs

in vitro.

85

The precise mechanisms by which Axl signalling and

func-tion respond to varying glucose levels remains unclear. However, these

findings may have important implications in terms of understanding the

pathological effects of chronic high blood glucose levels in diabetic

patients.

Vascular calcification, which is the process by which calcium builds up

within the vasculature, is particularly prevalent in advanced, inflammatory

atherosclerosis and correlates with worse clinical outcomes.

86–88

Conversely, absence of calcification in coronary arteries predicts a low

risk of CVD even in subjects with a high level of traditional risk factors.

89

During calcification, vascular pericytes undergo a process of osteogenic

differentiation, and Axl was identified as one of the genes which is

down-regulated when this process was explored in vitro.

90

Similarly, Axl

expression was shown to be down-regulated as cultured VSMCs calcify

their matrix, and Axl overexpression or activation inhibited calcification

in vitro.

91,92

More recently, miR-34a has been shown to promote VSMC

calcification in mice and in VSMCs in vitro, with the in vitro effect of

miR-34a showing a correlation with decreased Axl expression on the

cul-tured VSMCs.

93

Interestingly, work by another group has revealed that

the ability of hydroxy-3-methylglutaryl coenzyme A (HMG CoA)

reduc-tase inhibitors (statins) to prevent phosphate-induced calcification by

VSMCs in vitro occurs via restoration of the Gas6-Axl mediated survival

pathway.

94,95

Whether Axl affects vascular calcification in vivo has not yet

been determined.

Axl has also been reported to be expressed by cardiomyocytes.

36

In

patients with heart failure Axl levels are amplified both in terms of the

cardiac tissue and circulating soluble Axl (sAxl).

35

Furthermore, Gas6

and sAxl levels are found to increase in patients following ST-segment

el-evation MI

.

96

In both studies, the levels of Axl were predictive of adverse

pathology, such as the extent of left ventricular remodelling and of

fur-ther cardiovascular events. One study tested the effect of both KO and

cardiac-specific overexpression of Gas6 in a cardiac stress murine

mod-el.

97

They found that Gas6-deficient mice had decreased hypertrophy,

fi-brosis, and contractile dysfunction in the chronic stress overload

induced by aortic banding. Whereas cardiac-specific overexpression of

Gas6 enhanced these pathologies. This was ultimately attributed to Gas6

activation of the ERK signalling pathway, driving cardiac hypertrophy.

Interestingly, this process was reversed with the use of a

pharmacologi-cal inhibitor of ERK. As discussed previously, Gas6 activation of Axl can

induce the ERK signalling cascade,

98

which combined with evidence that

Axl levels are elevated in heart failure patients, points to the Gas6-Axl

axis as a potential novel therapeutic target in heart failure.

In summary, multiple studies suggest that GAS6/Axl drives

cardiovas-cular remodelling by regulating the biology of VSMCs, ECs,

cardiomyo-cytes, and potentially fibroblasts, thereby facilitating pathological

processes such as neointima formation, and remodelling in both the

heart and vasculature. Most importantly, Axl suppression can dampen

these adverse effects, suggesting possible therapeutic implications of

these studies.

3.2 Gas6-Axl and inflammation in CVD

Axl performs biphasic roles in the cells of the vasculature and immune

system. Axl expression has actually been suggested to drive

pro-inflammatory activation of VSMCs during vein-graft remodelling.

65

In this

study, vein-graft surgeries were performed to examine the effect of Axl

deficiency in both the vein graft donor and recipient mouse to compare

the effect of vascular vs. systemic expression of Axl. The authors found

that Axl depletion in all groups led to lower levels of MHC class II

ex-pression in the vein graft, indicating lesser immune activation. They also

observed down-regulation of various pro-inflammatory cytokines and

chemokines in Axl

-/-

SMCs compared with Axl

þ/þ

with and without

inter-feron gamma (IFN-c) stimulation. Remarkably, they found Axl deficiency

to increase the expression of SOCS1 in VSMCs, i.e., opposite to what is

observed in immune cells.

10,16,50

The transfer of Axl deficient haematopoietic myeloid cells to western

diet-fed low-density lipoprotein receptor KO (Ldlr

-/-

) mice had no effect

on atherosclerotic disease progression.

99

To date no studies have

exam-ined the effect of global Axl deficiency on atherosclerosis pathology, and

thus the effect of eliminating Axl expression in the vasculature has not

been addressed. This remains an important point for study, as Axl has

been shown to be present in human vessels with expression

down-regu-lated in atherosclerotic plaques compared with normal carotids.

100

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..

.

Furthermore, Axl expression is higher in vessels that are less prone to

develop atherosclerosis such as the left internal mammary artery

com-pared with the aorta.

101

Concomitantly, elevated serum levels of sAxl

have been detected in the setting of acute coronary syndromes.

102

The

Axl ligand Gas6 is expressed by ECs, VSMCs, and macrophages and its

expression increases with atherosclerosis development.

103,104

Genetic

KO

of Gas6 increases plaque stability in ApoE

-/-

mice, leading to increased

plaque content of SMCs and collagen and to reduced numbers of

macro-phages.

105

However, data from an epidemiological studies showed that

low levels of Gas6 correlate with increased risk of coronary artery

dis-ease (CAD) in patients with psoriasis.

106

An interesting study used chimeric mice with haematopoietic or

non-haematopoietic Axl deficiency to dissect the role of Axl expression in

immune vs. vasculature cells in hypertension.

67

The data indicated that

Axl-expressing immune cells drove pro-inflammatory gene expression

and increased immune cell infiltration in the kidney at early stages of

hy-pertension and that Axl expression in both immune and vascular cells

was detrimental in the later phases of hypertensive disease. Continuing

on from this, the same group demonstrated that Axl is critical for survival

of T lymphocytes, affecting vascular remodelling and inflammation in

DOCA-salt induced hypertension.

107

Axl has been suggested to influence natural killer (NK) cell

develop-ment. However, this conclusion is controversial, with studies supporting

a role for Axl in both the suppression and promotion of NK cells.

108–110

Although none of these studies are focused on cardiovascular disease,

this could be an interesting area of future study. On the one hand, NK

cells may have a potentially protective role in CAD based on strong

cor-relations between coronary heart disease and low NK levels in

humans.

111,112

However, animal studies suggest that NK cells are

athe-rogenic.

113

The role of Axl in NK cells and the role of NK cells

them-selves in cardiovascular disease represent areas that will benefit from

further studies, which is necessary for a better understanding of Axl’s

role in vascular pathology and its potential as a therapeutic target.

4. MerTK in CVD

4.1 MerTK-mediated efferocytosis in

atherosclerosis

MerTK deficiency leads to enhanced pathology in mouse models of

athe-rosclerosis.

30,32

One study backcrossed MerTK KOs into ApoE

-/-

mice,

which were maintained on western diet for either 10 or 16 weeks to

ex-amine the differential effect of MerTK deficiency on early or advanced

lesions.

32

The pathological effects of MerTK-deficiency were apparent in

advanced, as opposed to early, lesions owing to a marked decrease in

the clearance of apoptotic bodies and the subsequent accelerated plaque

necrosis. Interestingly, the study found no apparent effect on overall

le-sion size or plasma cholesterol profile, despite other studies showing

that effective efferocytosis is beneficial early on in lesion

devel-opment.

25–27

Thus, other phagocytic receptors may play a role in

effero-cytosis in early atherosclerosis, although Axl is not likely among these

receptors, as mentioned above, the transfer of Axl deficient bone

mar-row cells to Ldlr

-/-

mice had no effect on atherosclerotic disease

progression.

99

The effects of MerTK in atherosclerosis progression to date has been

investigated in phagocytic immune cells—primarily macrophages. This

can be inferred from a study which utilized a chimeric mouse model, in

which bone marrow cells deficient in MerTK were transferred into Ldlr

-/-mice.

30

Similar to the previous study, MerTK deficiency lead to

significantly higher levels of apoptotic debris accumulation within the

pla-que after 15 weeks of western diet. The authors were also able to show

increased inflammation in the lesions of these mice. In this study, there

was a 60% increased lesion size in the Mertk

-/-

Ldlr

-/-

mice compared with

Mertk

þ/þ

Ldlr

-/-

. It is possible the differences in effect on lesion size may

be due to differences between the mouse models and/or composition of

the diets used in the two studies. More recently, a macrophage Ca

2þ

/cal-modulin-dependent protein kinase IIc (CaMKIIc) pathway was shown to

play a key role in the development of necrotic atherosclerotic plaques

by preventing MerTK expression through the inhibition of the

transcrip-tion factors ATF6 and LXRa.

114

MerTK is expressed in macrophages in human atherosclerotic

arteries,

100

and MerTK induction is required for clearance of apoptotic

cells by human macrophages.

115

Interestingly, it has been suggested that

macrophage MerTK deficiency can occur near the necrotic cores of

hu-man plaques via the action of ADAM17-mediated MerTK cleavage.

55

ADAM17 can be activated by the byproducts of polyunsaturated fatty

acid oxidation and by inflammatory mediators, which are known to be

present within the necrotic core of atherosclerotic plaques.

53,55

Levels

of sMer within individual plaques were shown to correlate with the

extent of necrosis, and mice expressing genetically modified MerTK

re-sistant to cleavage (MerTK

D483-488

) showed improved lesional

efferocy-tosis, more stable plaques, and, interestingly, improved resolution of

inflammation

31

(see below).

Although research in this area has mainly focused on the role of

MerTK in macrophages, it should also be noted that brain microvascular

ECs have been shown to express MerTK,

116

and that it is required for

tightening the blood–brain barrier during viral infection.

117

In

atheroscle-rosis, inflammation and oxidative stress can destabilize the endothelial

barrier, contributing to pathology.

118

Therefore, MerTK, by maintaining

the integrity of the endothelial barrier, may in principle also contribute

to impeding the development of atherosclerosis; however, further work

needs to be done to explore this aspect.

4.2 MerTK-mediated efferocytosis in

myocardial infarction

Another important facet of cardiovascular pathology that relies on

effi-cient efferocytosis, includes the clearing of dead cardiomyocytes

follow-ing MI.

119–121

This process is orchestrated by various immune cells, and

the inflammatory profile of these cells can have a major influence on the

functional outcome and subsequent progression of heart failure.

122

MerTK-expressing monocyte/macrophages are key for the clearance of

injured cardiomyocytes and improve remodelling following MI in

mice.

123

Conversely, genetic deficiency of MerTK led to an increase in

the accumulation of apoptotic cardiac cells following experimental MI,

resulting in larger infarct sizes and worse cardiac functional outcomes.

124

Cardiac extracts from the Mertk

þ/þ

control mice showed the presence

of sMer following MI, which is likely due to the presence of post-MI

ADAM17-activating factors that promote MerTK cleavage.

125

In a study investigating adverse effects of post-MI

ischaemia–reperfu-sion (IR) in mice and humans using therapeutic interventions to restore

blood-flow, such as coronary stents and thrombolytic agents,

126,127

se-rum levels of sMer were found to be elevated.

56

In the mouse models,

this finding correlated with lower expression of intact MerTK on the

sur-face of cardiac macrophages. The study went on to show that mice

pos-sessing genetically modified cleavage-resistant MerTK (MerTK

D483-488

)

displayed improved levels of efferocytosis, reduced infarct size, and

im-proved cardiac function following IR. It was concluded that the crucial

(6)

..

.

role of MerTK in facilitating phagocytic clearance of cardiac debris

following MI was hindered by IR-induced MerTK cleavage. They

hypoth-esized that the trigger for this was recruitment of monocytes from the

circulation, which they tested by treating mice with an antagonist to

block C-C chemokine receptor type 2 (CCR2), a chemokine receptor

expressed by infiltrating circulatory monocytes, but not in

cardiac-resident macrophages. This treatment improved post-IR infarct size in

Mertk

þ/þ

but not Mertk

-/-

mice, suggesting that CCR2-mediated

infiltra-tion negatively affects ability of MerTK to effectively drive phagocytic

repair following IR. The exact mechanism for CCR2-dependent MerTK

cleavage is still ambiguous. One suggestion is activation of

ADAM17-mediated cleavage of MerTK by CCR2

þ

monocyte production of

ROS.

53,55,56

Interestingly, dying cardiomyocytes in the setting of MI promote

the release of MerTK from the surface of macrophages, which prevents

engulfment of dying cardiomyocytes.

125

The mechanism involves

up-regulation on cardiomyocytes of the ‘don’t-eat-me’ marker CD47

fol-lowing MI.

128

Although this process during MI is pathologic, as uncleared

apoptotic cardiomyocytes drive cardiac inflammation and further

cardio-myocyte death, it should be noted that in non-pathological conditions,

evading efferocytosis may preserve cardiomyocyte numbers in view of

their normally low regenerative capacity. In the pathologic setting of MI,

treatment with anti-CD47 subsequently enhanced cardiomyocyte

phagocytosis and reduced infarct size.

128

Accordingly, further

investiga-tion into the mechanism by which cardiomyocytes facilitate MerTK

shed-ding and how this can be prevented may present a novel target for

therapeutic intervention in improving post-MI recovery.

4.3 MerTK mediated resolution of

inflammation in atherosclerosis

The MerTK studies in the Apoe

-/-

and chimeric Ldlr

-/-

atherosclerosis

models found a heightened inflammatory pathology associated with loss

of MerTK.

30,32

Ait-Oufella et al.

showed that ex vivo cultured splenocytes

from Mertk

-/-

mice had increased production of pro-inflammatory

cyto-kines IFN-c, IL-12, and tumour necrosis factor (TNF)-a and decreased

production of anti-atherogenic IL-10, showing this phenotype to be

in-herent in these cells and not just a consequence of the defective

apopto-tic debris clearance. Mice expressing cleavage-resistant MerTK generate

increased levels of specialized pro-resolving mediators such as TGF-b

and IL-10 and lipid mediators such as resolvins in atherosclerotic

lesions,

31,37

which, together with enhanced efferocytosis, contribute to

attenuation of cardiovascular pathology in atherosclerosis. In terms of

lipid mediators, when MerTK engages a ligand, a particular tyrosine

resi-due in the cytoplasmic tail of MerTK signals inhibition of a calcium/

CaMKII pathway that normally favou

rs the biosynthesis of long-chain

un-saturated fatty acid-derived inflammatory mediators, notably

leuko-trienes, over resolution mediators such as lipoxins and resolvins.

37,62

Thus, MerTK signall

ing increases the ratio of

pro-resolving-to-pro-inflammatory lipid mediators.

Activation of LXR expression occurs as a result of apoptotic cell

clear-ance, particularly in the presence of excess-lipoprotein derived

choles-terol, as would be expected within advanced atherosclerotic lesions.

129

This process has been shown to promote expression of MerTK, which

mitigates pro-inflammatory cytokine release upon subsequent exposure

to cholesterol-loaded apoptotic macrophages.

24,130

In addition, human

protein S inhibits the expression of macrophage scavenger receptor A

through MerTK, leading to reduced uptake of modified lipoproteins.

131

Therefore, MerTK appears to sit at the interface between lipid

metabolism and inflammation within the plaque and functions to

attenu-ate inflammation in this environment.

5. Tyro3 in CVD

Apart from a study showing an association between Tyro3 and MerTK

variants and carotid atherosclerosis,

132

the net contribution of Tyro3 to

CVD has not been addressed to date. This may be because Tyro3 is

mainly localized to the central nervous system and reproductive organs,

and doesn’t show high levels of expression in the vasculature.

36,133,134

It’s ligand Pros1 has been found to correlate with coronary heart disease

risk and is expressed in atherosclerotic lesions.

100,135

Interestingly, Tyro3 is believed to negatively regulate T helper type 2

(Th2) cells via the suppression of a specific subset of CD11c

þ

DCs

expressing programmed cell death protein 2 (PDL2).

136

PDL2

þ

DCs are

associated with driving Type 2 (Th2-driven) immune responses, and

ex-pression of Tyro3 in these cells was shown to decrease their

Th2-associated molecule production. This pathway functions as part of a

neg-ative feedback loop, whereby Th2-associated cytokine IL-4 induces

sus-tained expression of Pros1, which then activated Tyro3-mediated

suppression of Th2 activation. In terms of relevance to CVD, while IL-4

and IL-5 have been shown to be atheroprotective,

137,138

IL-9 may be

pro-atherogenic.

139

IL-13 production exerts adverse effects on

cardio-vascular pathology by driving fibrosis of heart tissue in the context of

both ageing and IR injury,

140–142

but IL-13 secreted by regulatory T cells

may actually promote efferocytosis in atherosclerotic lesions.

143

These

combined observations provide a strong rationale for future studies on

the role of Tyro3 in CVD.

6. Conclusions and future questions

The TAM receptors—particularly MerTK and Axl—appear to have very

different roles in suppressing or driving elements of cardiovascular

pa-thology (Figure

2 ). In terms of what is currently known, MerTK is

funda-mentally protective in its role. This is mediated by distinct but

complementary functions: suppression of the inflammatory response,

enhancing the inflammation resolution response, and facilitating the

clearance of apoptotic cell debris. By these criteria, as well as the

protec-tive role of MerTK in the heart itself, enhancing MerTK synthesis or

func-tioning, and/or blocking its cleavage, may represent novel therapeutic

approaches to CVD. However, this approach must consider the possible

adverse effects of heightened MerTK function on cancer

144,145

and

possi-bly pathologic liver fibrosis.

146

There is controversy within the literature in terms of a protective vs. a

detrimental role of Axl in CVD. This largely stems from apparent

differ-ences in Axl function, particularly in terms of activating inflammation and

the cell type in which it is expressed. While there is a molecular signalling

pathway by which Axl has been shown to suppress the immune

re-sponse, the role of this anti-inflammatory pathway in vascular cells is

con-troversial.

16,65

On the other hand, Axl signall

ing may be protective

against vascular calcification.

90,91,95

Therefore, future work will be

re-quired to sort out the mechanisms that facilitate Axl’

s opposing roles in

vascular and immune cell types and how these roles affect overall

cardio-vascular pathology.

Tyro3 remains to be the least studied of the TAM receptors,

particu-larly in the area of cardiovascular disease. Other than one study which

found an association of a Tyro3 SNP in carotid atherosclerosis

132

(7)

..

we could find no published work to date investigating the role of Tyro3

in the context of cardiovascular pathology. However, Tyro3 may possess

a protective function in its ability to suppress of type 2 immune

responses, which promotes cardiac fibrosis.

140–142

Thus, with further

work in this area, it is possible that targeting Tyro could be considered as

a fibrosis-preventing therapy post-cardiac injury.

Finally, there are studies suggesting the Gas6/TAM signalling

pathway is essential for platelet activation and thrombus stabilization.

For example, mice deficient in Gas6 or TAM receptors or treated

with inhibitors of Gas6/TAM receptor pathways develop less arterial

and venous thrombosis. The GAS6/TAM receptor role in haemostasis

and thrombosis has been recently fully reviewed elsewhere

147

and is

therefore not the focus of the current review. Nonetheless, this

role of TAM receptors needs to be considered in view of the key

contribution of thrombosis to atherosclerosis and its clinical

complications.

Figure 2

Roles of TAM receptors in various cardiovascular diseases. Pathological roles for TAM receptor family members in cardiovascular disease are

shown in red, with protective roles depicted in green. MerTK-deficiency has been shown to be detrimental in atherosclerosis models owing to its ability to

dampen inflammation, promote resolution, and drive clearance of apoptotic bodies in the plaque necrotic core.

30,32,56,130

These processes can be inhibited by

MerTK cleavage, which occurs in necrotic, inflammatory plaques.

31,55,56

In hypertension models, Axl expression in both vascular and immune cells has been

im-plicated to drive pro-inflammatory responses in the kidney,

67

and to affect T cell survival, vascular inflammation and remodelling.

107

A major contribution to

heart failure in coronary heart disease is due to tissue damage and fibrosis following myocardial infarction. Efficient clearing of dead cardiomyocytes is crucial for

restoration of cardiac function, and MerTK has been shown to play a protective role in this setting.

124

This process can be hindered by cleavage of MerTK, which

is increased following ischaemia–reperfusion.

56

Although Tyro3 could potentially have a protective effect on the myocardium, as it suppresses Th2 responses

which drive cardiac fibrosis,

136,140–142

a direct causal link has not been shown to date. Gas6-Axl driven activation of the ERK signalling cascade in cardiomyocytes

is implemented in the pathological remodelling which occurs in heart failure patients.

35,96,97

Numerous studies have highlighted Axl to also have a pathological

role in vascular remodelling through increasing VSMC proliferation, migration, and immune activation, while also inhibiting VSMC apoptosis.

64,65,69,79

(8)

..

.

Conflict of interest: none declared.

Funding

This work was supported by the British Heart Foundation [PG/12/81/29897

to P.M., RE/13/5/30177, and FS/16/55/32731]; the Engineering and Physical

Sciences Research Council (EPSRC) [EP/L014165/1 to P.M.]; the European

Commission Marie Skłodowska-Curie Individual Fellowships [661369 to

P.M.]; the Arthritis Research UK [RACE20298 to M.K.S.]; and National

Institutes of Health [HL075662, HL127464, and HL132412 to I.T.].

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